Polynucleotide phosphorylase was used to covalently link poly(A) tracts having 5, 18, 31, 41, 59, 73, 100 and 150 residues to the 3' termini of E. coli 5S (3H) rRNA (5S (3H) rRNA (A)n). Upon hydrolysis of these polyadenylated polynucleotides by ribonucleases from Citrobacter sp., Enterobacter sp., bovine pancreas, human spleen and human plasma, it was found that the (A)n segment inhibited RNase activity, and that the inhibition was enhanced as the polyadenylic acid tract was lengthened. Certain polyamines, such as spermidine restores enzyme activity in the presence of 5S rRNA (A)n (N equals 73). Low concentrations of antimalarial compounds containing polyamine substructures, such as Atabrine (a 9-aminoacridine) or Plaquenil (a 4-aminoquinoline), also reverse (A)n induced inhibition of RNase activity while 8-aminoquinolines such as Primaquine were ineffective. Divalent metal ions (mg 2ion, Mu 2ion, Ni 2ion, or Co 2ion) also overcome inhibition, however, the action of metals may be distinguished from that of polyamines because in the absence of inhibition, 5S rRNA . (A)n is hydrolyzed at a faster rate than 5S rRNA only in the presence of metal ions. In all systems studied, inhibition of RNase activity mediated by 5SrRNA-(A)n is regulated by the chain length of the poly(A) segment, the concentrations of enzyme and substrate and the identity and concentration of metal ions or polyamines.